Alpha-alpha linking motifs and interhelical orientations

While the geometry and sequence preferences of turns that link two beta-strands have been exhaustively explored, the corresponding preferences for sequences that link helical structures have been less well studied. Here we examine the interhelical geometry of two connected helices as a function of their link's length. The interhelical geometry of a helical pair appears to be significantly influenced by the number of linking residues. Furthermore, for relatively short link lengths, a very limited number of predominant conformations are observed, which can be categorized by their phi/psi angles. No more than two predominant linking backbone conformations are observed for a given link length, and some linking backbone conformations correlate strongly with distinctive interhelical geometric parameters. In this study, sequence and hydrogen-bonding patterns were defined for predominant interhelical link motifs. These results should assist in both protein structure prediction and de novo protein design.     

MOMENT: software for analysis and display of amphiphilic regions in proteins

MOMENT is a 53 kbyte program written in Borland TurboPascalwith Borland Turbo Graphics, which will run on IBM PC-compatiblemachines. The program reads protein sequence files and calculatesthe hydrophobic moment of user-defined segments. The valuesproduced can be displayed in the form of a scan through thesequence, as vector diagrams and helical wheels, as plots ofhydrophobic moment versus hydrophobicity, or as hydrophobicmoment as a function of angle.     

Higher-order molecular packing in amyloid-like fibrils constructed with linear arrangements of hydrophobic and hydrogen-bonding side-chains

Various mutants of the protein fragment, barnase module-1 (1-24) were investigated in order to reveal the structural principle of amyloid-like fibrils. By means of circular dichroism spectroscopy, X-ray diffraction, electron microscopy, and thioflavin T binding assay, we found that the molecules containing two beta-strands and an intervening turn structure are assembled to form a cross-beta structure. Stabilization by both the hydrophobic interactions and hydrogen bonding between the respective paired side-chains on the coupled beta-strands was essential for fibril formation. These two types of interaction can also arrange the corresponding residues in lines on both sheet surfaces of protofilaments with a cross-beta structure. This leads to the most probable fibril structure constructed with the line-matching interactions between protofilaments. Consideration of the geometrical symmetry resulted in our finding that a limited number of essential models for molecular packing in fibril structure are stable, which would rationally explain the occurrence of two or three morphologies from an identical molecular species. The ribbon-like fibrils exhibited striped texture along the axis, which was assigned to a stacked two-sheet repeat as a structural unit. The comprehensively proposed structural model, that is, the sheet-sheet interaction between left-handed cross-beta structures, results in a slightly right-handed twist of beta-sheet stacking, which reasonably elucidates the intrinsic sizes of the fibril width and its helical period along the fibril axis, as the bias in the orientation of the hydrogen-bonded beta-strand pair at the lateral edge is larger than that at the central protofilament.     

Size and folding in globular proteins

We have modeled protein folding by packing a unified length of regular structural elements (alpha-helices and beta-sheets) into a 'cube'. In a globular protein with m alpha-helices and n beta-strands, this unified length is expressed in units of heptapeptides in alpha-helices, and in units of tripeptides in beta-strands. Calculations using published data show that a 4-helix bundle (m = 4, n = 0) has at least 2 x 2 x 2 helical heptapeptides; the 16-strand beta-barrel of porin (m = 0, n = 16) is at most 4 x 4 x 4 tripeptides in beta-strands. Compact, recurring protein modules with mixed helices and beta-strands are the ones that actually acquire a geometrically quasi-spherical, or cubic, shape.     

Describing protein structure: a general algorithm yielding complete helicoidal parameters and a unique overall axis

We present a general and mathematically rigorous algorithm which allows the helicoidal structure of a protein to be calculated starting from the atomic coordinates of its peptide backbone. This algorithm yields a unique curved axis which quantifies the folding of the backbone and a full set of helicoidal parameters describing the location of each peptide unit. The parameters obtained form a complete and independent set and can therefore be used for analyzing, comparing, or reconstructing protein backbone geometry. This algorithm has been implemented in a computer program named P-Curve. Several examples of its possible applications are discussed.     

A computer graphics program system for protein structure representation

We have developed a computer graphics program system for the schematic representation of several protein secondary structure analysis algorithms. The programs calculate the probability of occurrence of alpha-helix, beta-sheet and beta-turns by the method of Chou and Fasman and assign unique predicted structure to each residue using a novel conflict resolution algorithm based on maximum likelihood. A detailed structure map containing secondary structure, hydrophobicity, sequence identity, sequence numbering and the location of putative N-linked glycosylation sites is then produced. In addition, helical wheel diagrams and hydrophobic moment calculations can be performed to further analyze the properties of selected regions of the sequence. As they require only structure specification as input, the graphics programs can easily be adapted for use with other secondary structure prediction schemes. The use of these programs to analyze protein structure-function relationships is described and evaluated.     

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy¹ and two-dimensional (2D) electron crystallography² have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method³, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments⁴, microtubules⁵, amyloid fibers⁶, tobacco mosaic viruses⁷, and bacteria flagella⁸, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid⁹ is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.     

1H NMR assignments and secondary structure of human beta 2-microglobulin in solution.

Sequence-specific resonance assignments of human beta 2-microglobulin (M(r) 12,000) and its secondary structure are determined by 2D NMR techniques. The protein is found to contain two antiparallel beta-sheets each of four beta-strands with the beta-sheets being connected by a single disulfide linkage. No evidence for any regular helical structure is found. Amide proton-solvent-exchange rate constants and 3JHN alpha coupling constants are evaluated.     

Complexes of RecA with LexA and RecX differentiate between active and inactive RecA nucleoprotein filaments

The bacterial RecA protein has been the dominant model system for understanding homologous genetic recombination. Although a crystal structure of RecA was solved ten years ago, we still do not have a detailed understanding of how the helical filament formed by RecA on DNA catalyzes the recognition of homology and the exchange of strands between two DNA molecules. Recent structural and spectroscopic studies have suggested that subunits in the helical filament formed in the RecA crystal are rotated when compared to the active RecA-ATP-DNA filament. We examine RecA-DNA-ATP filaments complexed with LexA and RecX to shed more light on the active RecA filament. The LexA repressor and RecX, an inhibitor of RecA, both bind within the deep helical groove of the RecA filament. Residues on RecA that interact with LexA cannot be explained by the crystal filament, but can be properly positioned in an existing model for the active filament. We show that the strand exchange activity of RecA, which can be inhibited when RecX is present at very low stoichiometry, is due to RecX forming a block across the deep helical groove of the RecA filament, where strand exchange occurs. It has previously been shown that changes in the nucleotide bound to RecA are associated with large motions of RecA's C-terminal domain. Since RecX binds from the C-terminal domain of one subunit to the nucleotide-binding core of another subunit, a stabilization of RecA's C-terminal domain by RecX can likely explain the inhibition of RecA's ATPase activity by RecX.     

The effect of long-range interactions on the secondary structure formation of proteins

The influence of long-range residue interactions on defining secondary structure in a protein has long been discussed and is often cited as the current limitation to accurate secondary structure prediction. There are several experimental examples where a local sequence alone is not sufficient to determine its secondary structure, but a comprehensive survey on a large data set has not yet been done. Interestingly, some earlier studies denied the negative effect of long-range interactions on secondary structure prediction accuracy. Here, we have introduced the residue contact order (RCO), which directly indicates the separation of contacting residues in terms of the position in the sequence, and examined the relationship between the RCO and the prediction accuracy. A large data set of 2777 nonhomologous proteins was used in our analysis. Unlike previous studies, we do find that prediction accuracy drops as residues have contacts with more distant residues. Moreover, this negative correlation between the RCO and the prediction accuracy was found not only for beta-strands, but also for alpha-helices. The prediction accuracy of beta-strands is lower if residues have a high RCO or a low RCO, which corresponds to the situation that a beta-sheet is formed by beta-strands from different chains in a protein complex. The reason why the current study draws the opposite conclusion from the previous studies is examined. The implication for protein folding is also discussed.     

Protein Phosphatase 2A Is the Major Enzyme in Brain that Dephosphorylates τ Protein Phosphorylated by Proline-Directed Protein Kinases or Cyclic AMP-Dependent Protein Kinase

Abstract: The paired helical filament (PHF), which makes up the major fibrous component of the neurofibrillary lesions of Alzheimer's disease, is composed of hyperphosphorylated and abnormally phosphorylated microtubule-associated protein τ. Previous studies have identified serine and threonine residues phosphorylated in PHF-τ and have shown that τ can be phosphorylated at several of these sites by proline-directed protein kinases and cyclic AMP-dependent protein kinase. Here we have investigated which protein phosphatase activities can dephosphorylate recombinant τ phosphorylated with mitogen-activated protein kinase, glycogen synthase kinase-3β, neuronal cdc2-like kinase, or cyclic AMP-dependent protein kinase. We show that protein phosphatase 2A is by far the major protein phosphatase activity in brain that dephosphorylates τ phosphorylated in this manner.     

The structure of a filamentous bacteriophage

Many thin helical polymers, including bacterial pili and filamentous bacteriophage, have been seen as refractory to high-resolution studies by electron microscopy. Studies of the quaternary structure of such filaments have depended upon techniques such as modeling or X-ray fiber diffraction, given that direct visualization of the subunit organization has not been possible. We report the first image reconstruction of a filamentous virus, bacteriophage fd, by cryoelectron microscopy. Although these thin ( approximately 70 A in diameter) rather featureless filaments scatter weakly, we have been able to achieve a nominal resolution of approximately 8 A using an iterative helical reconstruction procedure. We show that two different conformations of the virus exist, and that in both states the subunits are packed differently than in conflicting models previously proposed on the basis of X-ray fiber diffraction or solid-state NMR studies. A significant fraction of the population of wild-type fd is either disordered or in multiple conformational states, while in the presence of the Y21M mutation, this heterogeneity is greatly reduced, consistent with previous observations. These results show that new computational approaches to helical reconstruction can greatly extend the ability to visualize heterogeneous protein polymers at a reasonably high resolution.     

Phasing statistics for alpha helical membrane protein structures

In this report we highlight the latest trends in phasing methods used to solve alpha helical membrane protein structures and analyze the use of heavy atom metals for the purpose of experimental phasing. Our results reveal that molecular replacement is emerging as the most successful method for phasing alpha helical membrane proteins, with the notable exception of the transporter family, where experimentally derived phase information still remains the most effective method. To facilitate selection of heavy atoms salts for experimental phasing an analysis of these was undertaken and indicates that organic mercury salts are still the most successful heavy atoms reagents. Interestingly the use of seleno‐l ‐methionine incorporated protein has increased since earlier studies into membrane protein phasing, so too the use of SAD and MAD as techniques for phase determination. Taken together this study provides a brief snapshot of phasing methods for alpha helical membrane proteins and suggests possible routes for heavy atom selection and phasing methods based on currently available data.     

Treponema pallidum TroA is a periplasmic zinc-binding protein with a helical backbone.

The crystal structure of recombinant TroA, a zinc-binding protein component of an ATP-binding cassette transport system in Treponema pallidum, was determined at a resolution of 1.8 A. The organization of the protein is largely similar to other periplasmic ligand-binding proteins (PLBP), in that two independent globular domains interact with each other to create a zinc-binding cleft between them. The structure has one bound zinc pentavalently coordinated to residues from both domains. Unlike previous PLBP structures that have an interdomain hinge composed of beta-strands, the N- and C-domains of TroA are linked by a single long backbone helix. This unique backbone helical conformation was possibly adopted to limit the hinge motion associated with ligand exchange.